PROJECT SUMMARY Eukaryotic cellular stress responses are highly conserved and precisely regulated. When these pathways malfunction there are often grave physiological consequences in the form of diseases such as cancer, neurodegenerative disease and autoimmune disorders. Ubiquitin-like modifications (UBLs) are rapid, reversible and can profoundly alter cell fate and function. Intriguingly, the majority of UBLs are involved in the cellular response to stress, in particular the response to infection, ER-stress and autophagy. Ubiquitin-like proteins share structural conservation with ubiquitin and also covalently modify target proteins, however UBLs have extremely divergent functions, which range from transcriptional repression to autophagy initiation. Unlike ubiquitin, the consequences of ISGylation or ATG12ylation on target protein fate and function are largely unknown. In this proposal, we aim to decode the post-translational landscape of the cell following eukaryotic stress responses using sophisticated proteomics strategies to address the fundamental question of how ISG15 and ATG12 alter the function of their covalent cellular targets. For ISG15, my laboratory?s recent work identified a novel role in the control of host metabolic processes and this proposal addresses how ISGylation of mTOR and other key upstream regulators of the catabolic process of autophagy tunes the metabolic and catabolic capacity of cells and animals. For ATG12, we have pioneered an in vivo genetic method coupled with cutting-edge proteomics to identify endogenous substrates of the autophagy-related ubiquitin-like protein ATG12 following induction of cellular stress pathways such as mitochondrial outer membrane permeabilization or endoplasmic reticulum stress.